Method for analyzing and selecting a specific droplet among a plurality of droplets and associated apparatus

ABSTRACT

The present invention relates to a method for analyzing and selecting a specific droplet among a plurality of droplets (4), comprising the following steps: —providing a plurality of droplets (4), —for a droplet (4) among the plurality of droplets, measuring at least two optical signals, each optical signal being representative of a light intensity spatial distribution in the droplet for an associated wavelength channel, —calculating a plurality of parameters from the optical signals, —determining a sorting class for a droplet according to calculated parameters, —sorting said droplet according to its sorting class, wherein the plurality of parameters comprises the coordinates of a maximum for each optical signal and a co-localization parameter and the at least two calculated parameters used for the determining step comprises the co-localization parameter.

The present invention concerns a method for analyzing and selecting aspecific droplet among a plurality of droplets.

In particular, the process is intended to screen and select the dropletsthat comprise a specific target element. For example, the specifictarget element can be the product of a biological reaction or a chemicalreaction.

The method is used to select microfluidic droplets. By “microfluidic”,it is generally meant that the dimensions of the passages in which thedroplets or fluid circulates are smaller than one millimeter and arecomprised for example between 1 μm and 1 mm.

Each droplet can be considered as a micro-container, wherein chemical orbiological reactions occur. They can be used for specific synthesis,screening of products or diagnosis.

In many assays, there is a need to sort droplets before the analysis inorder to enhance the efficiency of the assay. In other assays, there isa need to sort the droplets after a number of chemical, physical orbiological reactions in order to collect specific droplet content.

For example, to test in parallel the activity or the properties of thelarge number of variants of chemical or biological micro-reactors, it isknown to distribute the micro-reactors in droplets of an emulsion, thento conduct a chemical or biological reaction in each of themicro-reactors. It is then necessary to separate the droplets accordingto the product they contain, in particular to evaluate and isolate thereaction conditions and the micro-reactors having led to a significantreaction.

To isolate the droplets in which significant reaction has occurred, itis known to place selective fluorescent markers that are active when thesignificant reaction has occurred.

Then, the droplets are sorted manually or using automatic sortingmachines to separate those which reacted, for example, throughmicrofluidic or flow cytometry techniques. For example, a fluorescentactivated cell sorter (FACS) measures a fluorescence signal within thedroplets. Such techniques are relatively complex and expensive.

However, in specific assays, it is also important to differentiatedroplets based on more complex criteria. For example, it is important todistinguish droplets containing an aggregate of biological entities fromdroplets containing the same amount of single entities but notaggregated. Indeed, aggregation can provide risk of false positiveselection or false negative rejection in some tests.

One aim of the invention is therefore to provide a method for analyzingand selecting a specific droplet with a higher fidelity than existingsystems.

To this aim, the subject-matter of the invention is a method foranalyzing and selecting a specific droplet among a plurality ofdroplets, comprising the following steps:

-   -   providing a plurality of droplets,    -   for a droplet among the plurality of droplets, measuring at        least two optical signals, each optical signal being        representative of a light intensity spatial distribution in the        droplet for an associated wavelength channel,    -   calculating a plurality of parameters from the at least two        optical signals,    -   determining a sorting class for a droplet according to at least        two calculated parameters,    -   sorting said droplet according to its sorting class,        wherein the plurality of parameters comprises the coordinates of        a maximum for each optical signal and a co-localization        parameter and the at least two calculated parameters used for        the determining step comprises the co-localization parameter.

The method for analyzing and selecting a specific droplet among aplurality of droplets according to the invention may comprise one ormore of the following feature(s), taken solely, or according to anytechnical possible combinations:

-   -   a co-localization parameter is calculated by comparing the        position corresponding to the maximum intensity (as maximum peak        intensity or an integrated signal or ratio of between a maximum        value of an optical signal and an integration value of said        optical signal) of a first optical signal among the at least two        optical signals to the position corresponding to the maximum        intensity of a second optical signal among the at least two        optical signals; the colocalization parameter can be        rationalized by the size of the droplet, taking into account the        time interval between the maximum signal intensity of the at        least two different fluorescent channels. The value is thus        bounded in between 0 and 1 (or 0% and 100%), with a value of 1        (or 100%) being the perfect colocalization of the two maximum        intensity signal.    -   the plurality of parameters comprises at least one of the        following parameters:    -   a droplet width,    -   an integration of an optical signal,    -   a ratio between a maximum value of an optical signal and an        integration value of said optical signal,    -   the coordinates of a local maximum for an optical signal,    -   the number of local maxima in a droplet for an optical signal,    -   the calculation of the derivative of an optical signal and    -   the calculation of the second derivative for an optical signal;    -   a first optical signal among the at least two optical signals        comprises a plurality of local maxima, the plurality of        parameter comprises a multipeak co-localization parameter        calculated between the first optical signal and a second optical        signal comprising a local maximum, the multipeak co-localization        parameters being calculated with the following steps:    -   for each local maximum of the first optical signal, calculating        an intermediate co-localization parameter, by comparing the        position of the local maximum of the second signal to the        position of said local maximum of the first optical signal,    -   comparing the intermediate co-localization parameters, the        multipeak co-localization parameter being the lowest        intermediate co-localization parameter;    -   a co-localization parameter in a droplet is normalized by the        droplet width;    -   the step of measuring is performed for at least two droplets of        the plurality of droplets and the plurality of parameters        comprises the spacing between the two droplets; during the        determining step, at least a calculated parameter is compared to        predetermined threshold values;    -   during the measuring step, at least three optical signals are        measured, and wherein a plurality of co-localization parameters        are calculated by comparing the position of the maximum of the        optical signals two by two;    -   a method as previously described comprising the following step:    -   providing an apparatus comprising a channel adapted for a flow        of droplets, the apparatus comprising a detection area, and a        sorting area, the plurality of droplets circulating in the        channel,    -   carrying out a measurement for a droplet flowing in the        detection area;    -   a step of capturing a picture of the droplet during the        measuring step;    -   at least a droplet of the plurality of droplets comprises a        first element, the first element being fluorescent in a        wavelength channel associated to a first optical signal among        the at least two optical signals, and wherein at least a droplet        of the plurality of droplets comprises a second element, the        second element being fluorescent in a second wavelength channel        associated to a second optical signal among the at least two        optical signals;    -   the first and second element are chosen in the group of elements        consisting of a cell, a fluorescently labelled protein, a cell        labelling reagent, a fluorescently labeled antigen, a        fluorescently labelled antibody, a particle coated with a        biological entity, a nucleic acid, a peptide and a chemical        drug.

The invention also concerns an apparatus for analyzing and selecting aspecific droplet among a plurality of droplets comprising:

-   -   a detection assembly adapted to measure, for a droplet, at least        two optical signals, each optical signal being representative of        a light intensity spatial distribution in the droplet for an        associated wavelength channel,    -   a calculator for calculating a plurality of parameters from the        at least two optical signals,    -   a selecting unit for determining a sorting class for the droplet        according to at least two calculated parameters,    -   a sorting unit for sorting the droplet according to its sorting        class,        wherein the plurality of parameter comprises the coordinate of        the maximum for each optical signal and a co-localization        parameter and the at least two calculated parameters comprises        the co-localization parameter.

The apparatus according to the invention may comprise the followingfeatures:

-   -   the detection assembly comprises a light source and at least a        visible light sensitive detector.

The invention will be better understood, upon reading of the followingdescription, given solely as an example, and made in view of thefollowing drawings, in which:

FIG. 1 is a schematic view of an apparatus according to the invention;

FIG. 2 is a schematic summarizing the steps for a method according tothe invention,

FIG. 3 is an example of curve of measurement of an optical signal for asuccession of droplets;

FIG. 4 is an example of curves of measurement of two optical signals fora succession of droplets;

FIG. 5 is an example of curves of measurement of two optical signals fora droplet wherein a co-localization parameter calculation isillustrated,

FIG. 6 is an example of curves of measurement of two optical signals fora droplet wherein a multipeak co-localization parameter calculation isillustrated,

FIG. 7 is a schematic representation of a sample droplet used in abio-assay,

FIG. 8 is an example of dot plot representing the droplets according totwo parameters and a selecting gate,

FIG. 9 is an example of the dot plot representing the droplets accordingto selected co-localization parameter set above 90% confidence interval,for selecting co-localization events of soluble fluorescently labeledantigen and fluorescently labeled antibody,

FIG. 10 is an example of the dot plot representing the dropletsaccording to selected co-localization parameter set below 90% confidenceinterval, for excluding co-localizations events,

FIG. 11 is an example of the dot plot and droplet signal representingthe droplets according to selected co-localization parameter set above90% confidence interval, for selecting co-localization events of solublefluorescently labeled reporter cell and fluorescently labeled antibody,

FIG. 12 is an example of the dot plot and droplet signal representingthe droplet signal according to selected co-localization parameter setbelow 90% confidence interval, for excluding co-localizations events ofsoluble fluorescently labeled antibody producing cell and fluorescentlylabeled antibody,

FIG. 13 is an example of the dot plot and droplet signal representingthe droplets according to antibody/reporter cell co-localizationparameter set above 90% confidence interval and excluding antibodyproducing cell/reporter cell co-localization by setting the parameterbelow 90% confidence.

An apparatus 1 for analyzing and selecting a specific droplet among aplurality of droplets 4 according to the invention is shown in FIG. 1 .

The apparatus 1 comprises a droplet supply 6, a controller 8, a dropletssupport 10, a detection assembly 12, a calculator 14, a selecting unit16 and a sorting unit 18.

Advantageously the apparatus further comprises a monitor 20, with a manmachine interface. The droplet supply 6 is intended to provide aplurality of droplets 4 dispersed in a carrier fluid 22.

In the embodiment of FIG. 1 , the plurality of droplets 4 is asuccession of droplets 4.

The droplets 4 contain an inner fluid 24 immiscible with the carrierfluid 22. By “immiscible” it is generally meant that less than 0.01% ofthe inner fluid 24 is able to dissolve in the carrier fluid 22 at 25° C.and ambient pressure. For example, the inner fluid 24 is an aqueoussolution and the outer fluid 22 is a carrier oil.

For example, the inner fluid 24 contains at least a biological entityand a medium, which is loaded in the inner fluid 22 before forming eachdroplet 4. For example, the biological entity is a cell.

The content of the droplets 4 of the plurality of droplets 4 can bedifferent.

Advantageously, at least a droplet 4 of the plurality of droplets 4comprises a first element 26, being fluorescent in a first wavelengthchannel. At least a droplet 4 of the plurality of droplets 4 comprises asecond element 28, being fluorescent in a second wavelength channel.Each fluorescent element 26, 28 is characterized by an excitationspectrum and an emission spectrum.

The wavelength channels of the excitation maxima are usually separatedby at least 70 nm.

Advantageously, the first and second element 26, 28 are chosen in thegroup of elements consisting of a cell, a fluorescently labelledprotein, a cell labelling reagent, a fluorescently labeled antigen, afluorescently labelled antibody, a particle coated with a biologicalentity, a nucleic acid, a peptide and a chemical drug.

The particle can be a solid particle or a soft particle. For example,the particle is a magnetic particle, a colloidal particle, an hydrogelbead, a vesicle, a liposome, a droplet or other.

The second element is for example adapted to bind the first element. Forexample, the first element is a fluorescently labeled antigen and thesecond element is a fluorescently labelled antibody.

In reference to FIG. 1 , the droplet support 10 is a chip, onto which amicrofluidic pattern is formed. The droplets support 10 is preferablymade in one piece of a single material, in particular a polymericmaterial such as polydimethylsiloxane (PDMS) or polymethylmethacrylate(PMMA), polycarbonate (PC), epoxy, in particular photopolymerizableepoxy such as marketed by Norland Optical Adhesives (NOA) or glass.

A shown in FIG. 1 , the droplet support 10 comprises at least a workingchannel 30. The working channel 30 is connected upstream to the supply 6of the plurality of droplets 4 dispersed in the carrier fluid 22. Theworking channel 30 is emerging downstream into at least a sorting area32 of the sorting unit 18.

The working channel 30 is adapted for the measurement of an opticalsignal in the successive droplets 4.

The droplet support 10 defines a detection area 34 wherein the support10 is transparent in the wavelength channels used for the detection. Inthe detection area 34 the working channel is extending along alongitudinal axis X.

The dimension of the working channel 30 in the directions Y and Ztransversal relatively to the longitudinal axis X are adapted to thedimension of the droplets 4 such that the droplets 4 of the successionare passing one by one in the detection area 34.

The controller 8 is adapted to control the flowrate of the plurality ofdroplets 4 within the working channel 30. For example, the controller 8is connected to the droplet supply 6 to control the injection ofdroplets 4 and carrier fluid by the droplets supply 6. In addition, thecontroller 8 allows to control the spacing between droplets 4, thedetection time and the frequency of droplets 4 passing through thedetection area 34.

The detection assembly 12 is adapted to measure, for a droplet, at leasttwo optical signals, each optical signal being representative of a lightintensity spatial distribution in the droplet 4 for an associatedwavelength channel.

For example, the detection assembly 12 comprises, at least a lightsource 36 and at least a visible light sensitive detector 38. Forexample, the visible light sensitive detector 38 is a photomultiplier.

The light source 36 is adapted to illuminate the detection area 34. Forexample, the light source 36 is a white source exciting every visiblewavelength.

For example, the detection assembly 12 comprises a light source 36 foreach optical signal. Advantageously, a light source 36 is adapted toemit a light with a non-zero intensity in specific wavelength channelcorresponding to the fluorescence excitation spectrum of a fluorescentelement 26, 28 likely to be in at least a droplet 4 of the plurality ofdroplets. For example, the light source 36 is a laser. For example, thespecific wavelength channel is an excitation channel to allow thefluorescence of the first element 26 or the second element 28.

For example, the detection assembly 12 comprises a visible lightsensitive detector 38 for each optical signal. Each visible lightsensitive detector r 38 is adapted to record a voltage measurementcorresponding to the intensity of an optical signal emitted in thedetection area 34 according to the time.

Advantageously, each visible light sensitive detector 38 is sensitive toa specific wavelength channel corresponding to the fluorescence emissionspectrum of an element 26, 28 likely to be in at least a droplet 4 ofthe plurality of droplets 4. For example, the wavelength channelassociated to the first optical signal comprises the emission spectrumof the first element 26 and the wavelength channel associated to thesecond optical signal comprises the emission spectrum of the secondelement 28.

For example, the detection assembly is able to measure optically theintensity of an optical signal along a detection line D, extending alonga direction Y perpendicular to the longitudinal axis X of the workingchannel 30.

The optical signal measurement is taken on the dimension of the droplets4 when it is passing progressively in the detection area 34.

When the flowrate of the carrier fluid 22 and droplets 4 is known, ameasurement of the optical signal obtained on this detection line Dduring the time corresponds to a spatial scanning of the droplet 4crossing the detection line D.

The visible light sensitive detectors 38 are arranged to measure theirrespective optical signal simultaneously on the same detection line D.

Different measurements will be described in reference to FIG. 3 , FIG. 4, FIG. 5 and FIG. 6 , later in the description.

The detection assembly 12 is connected to the calculator 14.

The calculator 14 is adapted for calculating a plurality of parametersfrom the at least two optical signals. For example, the calculator 14comprises a memory and a real-time microprocessor.

The calculator 14 is adapted to retrieve, calculate, interpret thesignal in real-time according to the defined criteria.

The defined criteria are then loaded into the calculator unit 14 inorder to reduce the time of data transfer and calculation.

The calculator 14 is adapted to increase the throughput of the dataanalysis and sorting.

The memory comprises a plurality of software modules which can beexecuted to carry out the calculations of the parameters by theprocessor.

The plurality of parameter comprises the coordinate of the maximum foreach optical signal and a co-localization parameter and the at least twocalculated parameters comprises the co-localization parameter.

Different parameters and the method to calculate them will be describedlater in the description. The calculator is connected to the detectionassembly 12 and the selecting unit 16.

The selecting unit 16 is adapted for determining a sorting class for adroplet 4 according to at least two calculated parameters. For example,the selecting unit 16 comprises a memory and a microprocessor.

For example, the selecting unit 16 comprises a plurality of softwaremodules which can be executed to carry out to compare a calculatedparameter to a threshold value. The sorting criteria will be describedlater in the description.

The selecting unit 16 is connected to the calculator 14 and the sortingunit 18.

The sorting unit 18 is adapted for sorting the droplet 4 according totheir sorting class when the droplets 4 in different sorting area 32.For example, the sorting unit 18 comprises a different sorting area 32for each sorting class. Each sorting area 32 is connected to the workingchannel 30. Moreover, the sorting unit 18 comprises an orientating mean40 to orient the droplet 4 in each sorting area 32 according to thesorting class of the droplet 4. For example, the orientating mean 40comprises electrodes, or flow controller.

The monitor 20 is adapted to display of the measurements on graphs andto allow setting parameters for the calculation or the sorting criteria.

For example, the monitor 20 is adapted to display dot plots representingthe droplets 4 according to two different parameters. A dot plot isrepresented on FIG. 8 .

The monitor 20 is connected to the controller 8, the detection assembly12, the calculator 14, the selecting unit 16 and/or the sorting unit 18.

A method for analyzing and selecting a specific droplet 4 among aplurality of droplets 4 using the apparatus 1 will now be described inreference to FIG. 2 .

The method comprises a providing step 50, a measuring step 52, acalculating step 54, a determining step 56 and a sorting step 58.

During the providing step 50, a plurality of droplets 4 is provided atthe entrance of the working channel 30 by the supply 6.

The droplets 4 are circulated into the working channel 30, thecontroller 8 controlling the flowrate of the carrier fluid 22 anddroplets 4.

The droplets 4 arrive successively in the detection area 34 in front ofthe detection assembly 12.

During the measuring step 52, the droplet 4 in the detection area 34, isilluminated by the detection assembly 12 and at least two opticalsignals are measured by the detection assembly 12 while the droplet 4 ispassing through the detection area 34.

Each optical signal is representative of a light intensity spatialdistribution in the droplet for an associated wavelength channel.

During the calculating step 54, several parameters are calculated fromthe measured optical signals by the calculator 14.

In particular, during the calculating step 54, the calculator 14calculates a plurality of parameters from the at least two opticalsignals, wherein the plurality of parameters comprises the coordinatesof a maximum for each optical signal and a co-localization parameter.

Advantageously, the calculator 14 further calculates other parameters,as described hereafter.

For each optical signal taken alone, the calculator 14 can calculate:

-   -   a droplet 4 width,    -   the coordinates of a maximum for the optical signal,    -   the coordinates of a global maximum for the optical signal,    -   the coordinates of a local maximum for the optical signal,    -   the calculation of the derivative of the optical signal,    -   the calculation of the second derivative for the optical signal,    -   the number of local maxima in a droplet 4 for the optical        signal,    -   an integration of the optical signal,    -   a ratio between a maximum value of the optical signal and the        integration value of said optical signal.

Moreover, for a succession of droplets, for each optical signal takenalone the calculator 14 can calculate the distance between twosuccessive droplets.

In reference to the FIG. 3 , we will explain how these parameters arecalculated. In reference to FIGS. 4 and 5 , the calculation of aco-localization parameter will be explained. Then in reference to FIG. 6, the calculation of a multipeak co-localization parameter will beexplained.

The FIG. 3 shows an example of measurement obtained for a series ofsuccessive droplets 4 passing successively through the detection line Dfor one optical channel corresponding to a first wavelength channel. Forexample, this signal is measured on a first visible light sensitivedetector 38.

The time is represented in abscissas, and the intensity is representedin ordinates.

In the example of FIG. 3 , the curve 60 corresponds to the first opticalsignal.

For example, the first optical signal is measured in a green wavelength.The wavelength is for example associated to a first element 26 being alabelled CHO cell (Chinese hamster ovary cells) used in a test. Forexample, the CHO cells are stained with Calcein AM. The Calcein AM isknown to become a green fluorescent when digested by living cells. Thepresence of a high peak of fluorescence indicates that there is aliveCHO cell in the droplet 4.

The curve 60 is a continuous signal comprising a plurality of successivebell curves 62.

Each bell curve 62 corresponds to the emission collected by the visiblelight sensitive detector 38 from a different droplet 4 passing in thedetection area 34. There is a residual signal observed for each droplet4, for example, because the droplet 4 has a refractive index differentfrom the carrier phase 22. In alternative, the inner fluid 24 of thedroplet 4 comprises an autofluorescent medium that add a residualsignal.

The width w1 of the bell curve 62 measured at a predetermined intensitylevel 11, indicates the width of the associated droplet 4. The width ofthe droplet can be used for further calculation to normalize thedistance.

The baseline 64 between the bell curves 62 represents the emissioncollected by the visible light sensitive detector 36 by the carrierphase 22 between two successive droplets. It has a lower intensity thanthe signal measured within the droplet 4.

The distance d between the centers of two successive bell curves 62corresponds to the spacing between the two successive droplets 4 alongthe X axis.

On the FIG. 3 , only some droplets 4 present a maximum peak 68distinguishable from the bell curve 62. In the example, those droplets 4correspond to droplets 4 containing an alive CHO cell.

For example, to determine the coordinate of the maximum, the opticalsignal is approximated by an interpolation function by the calculator14. Then the first derivate of the function is calculated by thecalculator. The second derivate of the function is calculated by thecalculator. The second derivate indicates the curvature of the function.A local maximum is a point where the first derivate is zero and thesecond derivate is negative.

The coordinates of the maximum peak 68 are memorized by the calculator.

In some assays, the intensity of the maximum peak can, for example, beused to calculate the concentration of the associated element in thedroplet. The position of the maximum peak is used for the determinationof co-localization parameters as it will be explained below in referenceto FIGS. 4, 5 and 6 .

The calculator 14 comprises several filters to avoid false local maximumdetection that are due to noise. The filters are based on value of peakwidth threshold, peak height threshold or peak excursion criteria. Thesevalues can be predetermined or settled by the user.

A global maximum is the highest maximum value as a function ofintensity.

Moreover, as this signal is continuous it is possible to integrate thissignal for each bell curve 62. For example, the signal is integratedbetween two threshold lines 70, 72 represented in the FIG. 3 . Forexample, the threshold lines 70, 72 are predetermined by the usermanually or automatically by a software module. In alternative or inaddition, the value can be changed manually by the user via the monitor20.

This integration value can be used for further calculation to normalizethe intensity measured. For example, the calculator can calculate aratio between a maximum value of the optical signal and the integrationvalue of said optical signal.

On FIG. 3 , a droplet 74 presents two maximum peaks. In the example, thedroplet 74 contains two alive CHO cells. In those droplets 4 two maximumcoordinates are calculated.

The detection of multi-peak and calculation of their coordinates andarea is particularly advantageous to detect elements for which theloading is dependent on a Poisson distribution, such as particles. Theparticles are for example cells. Some droplets 4 contain no particles,some droplets 4 contains only one particle, and the other more than oneparticle. The determination of the number of maximum peak for a channelallows knowing the number of particles associated with this wavelengthin the droplet. It can be particularly interesting for assays whereinthe results are dependent on the number of particles. It is particularlyimportant to distinguish these droplets 4 for single cell assays.

FIG. 4 shows another example of measurement obtained for a series ofsuccessive droplets 4 passing successively through the detection line Dfor two optical channels corresponding to a first wavelength channel anda second wavelength channel.

Each optical signal is represented with a different curve 80, 82. Forexample, the curve 80 represented with a continuous line is measured ona first visible light sensitive detector 38 and corresponds to the firstoptical signal associated to a first element 26. The curve 82represented in dotted lines on FIG. 4 is measured on a second visiblelight sensitive detector 38 and corresponds to the second opticalsignal, associated to a second element 28. Each signal is measured in adifferent fluorescence channel.

The calculator can calculate for each optical signal, the parametersdescribed above in reference to FIG. 3 .

Moreover, by comparing the two optical signals, the calculatorcalculates a co-localization parameter. The calculation is explained inmore detail by reference to FIG. 5 .

FIG. 5 illustrates the measurement for one droplet for the two signalsin more details.

The calculator calculates the coordinates of a maximum 84, 86 for eachoptical signal in the droplet.

Then, the calculator 14 calculates the distance Δ between the positioncorresponding to the maximum intensity of the first optical signal andthe position corresponding to the maximum intensity of the secondoptical signal. This distance Δ is a co-localization parameter.

The lower the distance Δ is, the more the elements 26, 28 associated tothe optical wavelength are close.

Advantageously, the calculator normalizes the co-localization parameterby the droplet width.

After the normalization, if the co-localization between the elements isideal the co-localization parameter is equal to 1. After thenormalization, if there is no co-localization, the co-localizationparameter is equal to 0.

A co-localization parameter is useful to detect binding between twoelements 26, 28. Indeed, for example if a fluorescent antigen is bondedto a fluorescent antibody in a droplet, the co-localization parameterassociated to the signal of the antigen and the signal of the antibodywill be high.

The co-localization parameter can be represented as a dot plot format asa function of the max. peak of the dropcode (V) or any other parameter(droplet width, second co-localization parameter). Typical example, isshown in FIG. 9 , where a confidence interval is defined 117. In someembodiment the confidence interval is defined for targeting the highestprobability 119 of true co-localization of two events. In the givenexample, the confidence interval (determining labelled-antigen andlabelled secreted antibody), is advantageously set between 90% and 100%,in order to precisely select the droplets having signal with the minimaldistance d between the antigen (peak1) 122, 120 and the antibody (peak2)122, 121. According to a preferred embodiment of the present invention,the co-localization parameter (A) is selected by having a confidenceinterval ranging from 90% to 100%.

Another application, described in FIG. 10 , is selection ofco-localization event of fluorescently labelled antibody 123 binding tofluorescently labelled reporter cells 122.

In some other embodiment the co-localization parameter is used as anexclusion criterion. The confidence interval represented in FIG. 10 isset to specifically select the droplets below the 90% of probability fortwo peaks being co-localized 118. This feature is particularly importantfor excluding non-specific binding events. Typical example includes casewhere fluorescent antibody 123, 120 is co-localized with fluorescentlylabelled antibody producing cells 124, 121.

In some other embodiment, described in FIG. 13 , the co-localizationparameters are used in combinations or exclusion modes. Typical exampleis the exclusion of false positive hit and/or fine selection of givendroplet population of interest. In a given example, droplets areselected if two signals co-localized but a third one has to be excluded.Typical case is where fluorescent antibody 123, 120 is co-localized withfluorescently labelled antibody producing cell but not on fluorescentlylabelled reporter cells 126, these two co-localization events are thusexcluded. Another typical case is where fluorescent antibody 123, 120 isco-localized with fluorescently labelled reporter cells 124, 121 andshould not co-localize with fluorescently labelled antibody producing126, 127.

In another example during the measuring step, at least three opticalsignals are measured, and a plurality of co-localization parameters iscalculated by comparing the position of the maximum of the opticalsignals two by two.

FIG. 6 shows another example of measurement obtained for a uniquedroplet 4 passing through the detection line D for two optical channelscorresponding to a first wavelength channel and a second wavelengthchannel.

This example is for illustrating the calculation of multipeakco-localization parameter between a first optical signal and a secondoptical signal, one of them comprising a plurality of local maxima.

Each optical signal is represented with a different curve 90, 92. Forexample, the curve 90 represented with a continuous line is measured ona first visible light sensitive detector 38 and corresponds to the firstoptical signal associated to a first element 26. The curve 92represented in dotted lines on FIG. 4 is measured on a second visiblelight sensitive detector 38 and corresponds to the second opticalsignal, associated to a second element 28. Each signal is measured in adifferent fluorescence channel.

In this example, the first optical signal comprises three local maxima94, 96, 98 and the second optical signal comprises a local maximum 100.

The calculator calculates the coordinates of the plurality of localmaxima 94, 96, 98 for the first optical signal and the coordinates ofthe local maximum 100 of the second optical signal.

The calculator calculates the multipeak co-localization parameterbetween the first optical signal and a second optical signal, with thefollowing steps:

-   -   for each local maximum 94, 96, 98 of the first optical signal,        the calculator calculates an intermediate co-localization        parameter d1, d2, d3, by comparing the position of the local        maximum 100 of the second signal to the position of said local        maximum 94, 96, 98 of the first optical signal,    -   then the calculator 14 compares the intermediate co-localization        parameters d1, d2, d3, the multipeak co-localization parameter A        being the lowest intermediate co-localization parameter.

In the example, three intermediate co-localization parameters d1, d2,d3, are calculated. It appears that the central local maximum 96 is theclosest to the local maximum 100 of the second optical signal. Themultipeak co-localization parameter A is the intermediateco-localization parameter d2 calculated between the central localmaximum 94 and the local maximum 100 of the second optical signal.

Advantageously, during the calculating step 54, the calculatedparameters are stored in a memory for the determining step 56 and/or forfurther utilization.

During the determining step 56, the selecting unit 16 decides a sortingclass for a droplet 4 according to at least two calculated parameters,comprising at least a co-localization parameter.

The number of sorting class depends on the assay and the possibility ofthe sorting unit. There is at least two sorting class.

For example, a sorting class is a class of droplets 4 to keep. Forexample, a sorting class is a class of droplets 4 to exclude. Forexample, a sorting class is a class of sample droplets; a sorting classis a class of positive control droplets 4 or a class of negative controldroplets.

Advantageously, during the determining step 56, the calculatedparameters are compared to selection criteria or threshold values.Advantageously, during the determining step, at least a calculatedparameter is compared to predetermined threshold values. In alternativeor in addition, some thresholds are determined manually by a user viathe monitor. The criteria used in the determining step 56 can be adaptedto the assay.

For example, for a selection on two parameters, a dot plot isrepresented on the screen of the monitor 20. To fix the thresholds fortwo specific parameters simultaneously, the user can draw a selectinggate around the selected or excluded droplets 4 in the associated dotplot via a human machine interface as illustrated on FIG. 8 .

For example, the determining step 56 comprises several steps eachselection step corresponding to a selection based on different criteria.Each parameter calculated by the calculator can be used for theselection. The sorting class of a droplet is attributed after eachselection step planned for the assay.

Advantageously, the user can change the number and type of selectionsteps via the monitor 20. In alternative, the number and type ofselection steps are memorized in the selecting unit 16 for a type ofassay.

Advantageously, the calculating step 54 and the determining step 56 canbe performed in parallel.

For example, the calculator 14 will stop performing calculation onexcluded droplets. It helps the method to be more rapid by avoidinguseless calculation.

In alternative all calculation steps are performed before thedeterminations steps.

Some example of selection criteria will be described hereinafter.

For example, in a step the selecting unit limits the population ofdroplets 4 to droplets 4 with a high co-localization between twoelements 26, 28 in the droplet 4. This selection is based on theco-localization parameters between the two signals associated to therespective element 26, 28. This selection is useful for example toselect droplets 4 where a binding between the two elements 26, 28occurs.

In alternative or in addition, the selecting unit rejects from theselected population the droplets 4 with a high co-localization betweentwo elements in a droplet. This selection is useful for example toreject droplets 4 where there is a binding between the two elements. Forexample, such a rejection is useful to exclude droplets 4 containingaggregates of cells. For example, if an antibody is bound on the surfaceof the secreting cell, it will be difficult to analyze the antibodyspecificity for an antigen.

For example, in a step, the selecting unit 16 limits the population ofdroplets 4 to droplets 4 with a correct width.

For example, in a step, the selecting unit limits the population ofdroplets 4 to droplets 4 containing an element. This selection is basedon the intensity value of the global maximum peak for the associatedsignal and on the ratio between the maximum value of the optical signaland the integration value of said optical signal. For example, thedroplets 4 which are in a threshold gate for the intensity value of theglobal maximum peak for the associated signal and the ratio between themaximum value of the optical signal and the integration value of saidoptical signal are kept.

Then during the sorting step 58, the sorting unit 18 sorts the droplets4 according to their sorting class. Each droplet 4 is oriented to asorting area 32 associated to its sorting class.

It is then possible to collect the droplets 4 or their content forfurther reaction or analysis.

Furthermore, advantageously, the optical signals, each parametercalculated and/or each sorting criteria are memorized. Therefore, it ispossible to use these data for further analysis.

Furthermore, the method advantageously comprises the step of capturing apicture of the droplet 4 during the measuring step. For example, thepicture is a snapshot of the sorted droplet 4. For example, the pictureis a one dimensional plot of the droplet 4 of interest.

A more specific example of application will now be described toillustrate the advantages of the invention.

The following example illustrates the droplets 4 can be sorted accordingto several criteria. The example is illustrated by the FIGS. 7 and 8 .

The goal of this assay is to recover specifically droplets 4 withantibody producting cells 110 able to produce an antibody 112 that canbind to a surface target 114 of a CHO (Chinese hamster ovary) cell 116.Such a droplet is schematically represented on FIG. 7 . It is thereforenecessary to recover the droplets 4 where the antibody 112 signalco-localizes with the target 112 but not with the B cells 116, thedroplet containing both a CHO cell 114 and a B cell 110.

In the assay, the CHO cells are stained with Calcein AM. The CHO cellused for the assay comprises at their surface a target antigen. The Bcells are stained with Calcein AM Violet.

Every droplets 4 of the assay comprises a droplet staining such assulforhodamine B, and a labelled antibody detection reagent, forinstance an anti-mouse IgG Fc AlexaFluor647.

In the example of FIGS. 7 and 8 , the succession of droplets 4 comprisesa plurality of positive control droplets, then a plurality of negativecontrol droplets, and finally a plurality of sample droplets.

The calculator 14 associates a drop code to each droplet 4 depending onthe order where it passes in the detection area 34. A predefined dropcode corresponds to the droplet of the plurality of sample droplets.

The positive control droplets 4 comprise of a CHO cell and an antibodyknown to be able to bind the target. The negative control droplets 4comprising of an aqueous medium, but do not comprise a CHO cell nor a Bcell.

Four optical signals are measured by the detection assemblysimultaneously.

For simplicity of explanation, the optical signal associated to the CHOcalcein AM stain is called green signal, the optical signal associatedto the B cells calcein AM violet stain is called violet signal. Theoptical signal associated to the drop code is called orange signal. Theoptical signal associated to the antibody binding detection reagent iscalled red signal.

From the orange signal, the calculator calculates the droplets 4 widths.From the red signal, the calculator calculates the coordinates of thelocal maxima, called hereinafter binding maxima. From the violet signal,the calculator calculates the coordinates of the local maxima, calledhereinafter B cell maxima. From the green signal, the calculatorcalculates the coordinates of the local maxima, called herein after CHOmaxima.

During the determination step, every droplet presenting a droplet widthhigher than a specific threshold or lower than another specificthreshold is rejected by the selecting unit. With these criteria, signaldue to impurities or droplets 4 difficult to screen and analyze becauseof their dimension are not kept. These droplets 4 and impurities cancome from emulsion instability or the spontaneous coalescence of aplurality of successive droplets 4.

During the determination step, among the remaining droplets, everydroplet presenting, for the red signal, corresponding to the antibodybinding reagent, an intensity for a binding maximum higher than athreshold which is associated with a sorting class to keep and the otherare associated to a class to separate. For example, the threshold is 0.1in an arbitrary unit based on background fluorescence and the positivecontrol droplets 4. After this step the negative control droplets 4 arein the class to separate and the positive control droplets are in thesorting class. The sample droplets 4 can be in the sorting class or inthe class to separate, but only the sample droplets 4 being in thesorting class can be selected as positive at the end of the determiningstep. With this criterion, every droplet containing the antibody that isspecific to the target is kept in the sorting class. In an example, withthis criterion only 0.16% of sample droplets 4 were kept in this sortingclass.

For the violet signal, corresponding to the B cells, the selecting unit16 associates every droplet presenting a maximal intensity under aspecific threshold to another class to separate. In complement or inalternative, the selection is made by a dot plot gating as representedin FIG. 8 . The dot plot represents the droplets 4 according to theintensity of B cells maxima and to the drop code. The gate includes thedroplets 4 with a low drop code and with a B cell maximum intensitycomprised between 0.01 V and 5 V. With this criterion, the droplets 4without B cells are excluded. For example, after this selection only 17%of droplets 4 are kept in the sorting class.

For the green signal, corresponding to alive CHO cells, every droplet inthe class to sort presenting a maximal intensity under a specificthreshold are associated to another class to separate. The dot plotrepresents the droplets 4 according to the intensity of CHO cells maximaand to the drop code. The gate includes the droplets 4 with a low dropcode corresponding to the sample series and with a CHO cell maximumintensity comprises between 0.01 V and 5 V. With this criterion, thedroplets 4 without CHO cells are not kept. For example, with thisselection only 70% of droplets 4 remains in the sorting class.

In alternative or in complement, the selection on the violet signal andthe green signal are made simultaneously. A dot plot representing everydroplet according to the CHO maximum intensity and the B cell maximumintensity is displayed on the monitor. The gating is made such thatevery droplet 4 kept in the class to sort has both a CHO cell and a Bcell.

After that the co-localization parameter between the green signal,corresponding to the CHO cells and the red signal corresponding to thebinding are calculated for the remaining droplets.

Furthermore, the co-localization parameter between the violet signal,corresponding to the B cells and the red signal corresponding to thebinding are calculated for the remaining droplets. Furthermore, theco-localization parameter between the violet signal, corresponding tothe B cells and the green signal, corresponding to the CHO cells arecalculated for the remaining droplets.

Then during a determining step 56, the droplets 4 with a highco-localization parameter between the red signal and CHO cells are keptin the sorting class.

Then the droplets 4 with a high co-localization parameter between thedetection reagent and B cells are rejected.

Finally, the droplets 4 with a high co-localization parameter betweenthe CHO cells and B cells are rejected. In this example, if the CHO cellco-localizing with a binding reagent is also co-localizing with a Bcell, the droplets 4 are excluded because it can be a false positive.Indeed, the B cells may have secreted antibody that are not bound to thetarget but detected by the binding agent.

This leads to a specific population of droplets 4 in the sorting classcomprising exclusively droplets 4 with B cells able to produce anantibody that can bind to a surface target of a CHO cell.

Then the droplets 4 were sorted based on their sorting class.

At each step of selection, the excluded droplet can be associated to adifferent sorting class. It allows performing several analyses on thedroplets 4. For example, the positive control droplets 4 can berecovered for the following analysis.

The invention provides a method for analyzing and selecting a specificdroplet with a higher fidelity than existing systems. Indeed, it ispossible to sort the droplets 4 according to multiple criteria. Theco-localization parameters combined with other parameters are useful toanalyze the spatial relative positions of elements, which can have aninfluence on assay results.

In another embodiment, the plurality of droplets 4 is an emulsion. Thedroplets 4 are stored in a microfluidic chamber, wherein the measurementstep is performed. The detection assembly 12 is adapted to scanspatially each droplet 4 in the chamber so as to measure the lightintensity distribution for a wavelength channel.

1. A method for analyzing and selecting a specific droplet among aplurality of droplets (4), comprising the following steps: providing aplurality of droplets (4), for a droplet (4) among the plurality ofdroplets, measuring at least two optical signals, each optical signalbeing representative of a light intensity spatial distribution in thedroplet (4) for an associated wavelength channel, calculating aplurality of parameters from the at least two optical signals,determining a sorting class for a droplet according to at least twocalculated parameters, sorting said droplet according to its sortingclass, wherein the plurality of parameters comprises the coordinates ofa maximum for each optical signal and a co-localization parameter (Δ)and the at least two calculated parameters used for the determining stepcomprises the co-localization parameter (Δ).
 2. A method according toclaim 1, wherein a co-localization parameter (Δ) is calculated bycomparing the position corresponding to the maximum intensity of a firstoptical signal among the at least two optical signals to the positioncorresponding to the maximum intensity of a second optical signal amongthe at least two optical signals.
 3. A method according to any of theclaims 1 and 2, wherein the co-localization parameter (Δ) is selected byhaving a confidence interval ranging from 90% to 100%.
 4. A methodaccording to any of the claims 1 to 3, wherein the plurality ofparameters comprises at least one of the following parameters: a droplet(4) width, an integration of an optical signal, a ratio between amaximum value of an optical signal and an integration value of saidoptical signal, the coordinates of a local maximum for an opticalsignal, the number of local maxima in a droplet for an optical signal,the calculation of the derivative of an optical signal and thecalculation of the second derivative for an optical signal.
 5. A methodaccording to any of the preceding claims, wherein a first optical signalamong the at least two optical signals comprises a plurality of localmaxima, the plurality of parameter comprises a multipeak co-localizationparameter calculated between the first optical signal and a secondoptical signal comprising a local maximum, the multipeak co-localizationparameters being calculated with the following steps: for each localmaximum of the first optical signal, calculating an intermediateco-localization parameter, by comparing the position of the localmaximum of the second signal to the position of said local maximum ofthe first optical signal, comparing the intermediate co-localizationparameters, the multipeak co-localization parameter being the lowestintermediate co-localization parameter.
 6. A method according to any ofthe preceding claims, wherein a co-localization parameter in a droplet(6) is normalized by the droplet width.
 7. A method according to any ofthe preceding claims, wherein the step of measuring is performed for atleast two droplets (4) of the plurality of droplets (4) and theplurality of parameters comprises the spacing between the two droplets(4).
 8. A method according to any of the preceding claims wherein duringthe determining step, at least a calculated parameter is compared topredetermined threshold values.
 9. A method according to any of thepreceding claims, wherein, during the measuring step, at least threeoptical signals are measured, and wherein a plurality of co-localizationparameters are calculated by comparing the position of the maximum ofthe optical signals two by two.
 10. A method according to any of thepreceding claims further comprising the following step: providing anapparatus (1) comprising a channel (30) adapted for a flow of droplets(4), the apparatus (1) comprising a detection area (34), and a sortingarea (32), the plurality of droplets (4) circulating in the channel(30), carrying out a measurement for a droplet (4) flowing in thedetection area (34).
 11. A method according to any of the precedingclaims comprising a step of capturing a picture of the droplet (4)during the measuring step.
 12. A method according to any of thepreceding claims wherein at least a droplet (6) of the plurality ofdroplets (4) comprises a first element (26), the first element (26)being fluorescent in a wavelength channel associated to a first opticalsignal among the at least two optical signals, and wherein at least adroplet (4) of the plurality of droplets (4) comprises a second element(28), the second element (28) being fluorescent in a second wavelengthchannel associated to a second optical signal among the at least twooptical signals.
 13. A method according to claim 12, wherein the firstand second element (26, 28) are chosen in the group of elementsconsisting of a cell, a fluorescently labelled protein, a cell labellingreagent, a fluorescently labeled antigen, a fluorescently labelledantibody, a particle coated with a biological entity, a nucleic acid, apeptide and a chemical drug.
 14. An apparatus (1) for analyzing andselecting a specific droplet (4) among a plurality of droplets (4)comprising: a detection assembly (12) adapted to measure, for a droplet(4), at least two optical signals, each optical signal beingrepresentative of a light intensity spatial distribution in the droplet(4) for an associated wavelength channel, a calculator (14) forcalculating a plurality of parameters from the at least two opticalsignals, a selecting unit (16) for determining a sorting class for thedroplet (4) according to at least two calculated parameters, a sortingunit (18) for sorting the droplet (4) according to its sorting class,wherein the plurality of parameter comprises the coordinate of themaximum for each optical signal and a co-localization parameter and theat least two calculated parameters comprises the co-localizationparameter.
 15. An apparatus (1) according to claim 14, wherein thedetection assembly comprises a light source (36) and at least a visiblelight sensitive detector (38).